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PubMed1984

Generation of chemotactic activity in serum by Haemophilus influenzae type b.

Studies were performed to characterize chemotactic activity generated by Haemophilus influenzae type b (HiTb) in serum or elaborated independent of serum. Neutrophil aggregometry, Sephadex G-75 gel chromatography, and anti-C5 neutralization studies were used to demonstrate that the complement fragment C5a represented the major chemotactic moiety derived from HiTb-serum interactions. HiTb elaborated minimal chemotactic activity independently. Maximal C5a generation by HiTb as measured by neutrophil response in chemotaxis, shape change, and aggregation assays required specific antibody to the capsular polysaccharide, polyribosyl ribitol phosphate (PRP). Significantly more C5a was generated in pooled normal human serum containing high titers of anti-PRP (determined by an enzyme-linked immunosorbent assay) than in hypogammaglobulinemic serum. Furthermore, C5a generated in hypogammaglobulinemic serum reconstituted with purified high-titer immunoglobulin G, hyperimmune rabbit serum or heat-inactivated normal human serum was comparable to that generated in normal human serum. Absorption of antibody with PRP versus whole HiTb showed a contribution by non-PRP-directed antibody. As shown with the use of C4-deficient guinea pig serum, C5a generation occurred via the alternative complement pathway in nonimmune serum, and activation of the alternative complement pathway was facilitated by specific anti-PRP. C5a generation in test sera was proportional to its opsonic activity for HiTb as assessed by a luminol-chemiluminescence assay. Overall low levels of C5a activity were observed in 13 pediatric patient serum samples obtained during the acute phase of HiTb meningitis, and no pulmonary symptoms or radiographic abnormalities consistent with a leukocyte aggregation syndrome were observed in these patients.

Antibodies, Bacterial↗
PubMed1984

Activation of complement system by porins extracted from Salmonella typhimurium.

The effect of porins purified from Salmonella typhimurium on the complement system was investigated both in vitro and in vivo. Incubation of porins with either human or guinea pig serum resulted in the consumption of the total complement activity when an amount of porins ranging from 8 to 10 micrograms per 100 microliters of serum was used. The activation of the complement system was temperature dependent, suggesting an active process rather than passive adsorption of the complement components by porins. In addition, the activation had a fast kinetic and proceeded mainly through the classical pathway. This conclusion is supported by the consumption of C1s and C4 in normal human serum treated with porins and also by the depletion of C3 activity in the C1s-deficient serum which was marked only when purified C1s was added to the serum before incubation with porins. Injection of 100 micrograms of porins into guinea pigs induced profound complement consumption at 6 h postinjection that persisted up to 12 h. We conclude from this study that porins can effectively contribute to complement activation and to subsequent biological events induced by gram-negative bacteria.

Animals↗
PubMed1986

Modulation of complement fixation and the phlogistic capacity of group A, B, and D streptococci by human lysozyme acting on their cell walls.

Streptococci and streptococcal cell wall fragments induce arthritis in rats, with the severity and duration depending on the capacity of the cells or cell fragments to resist degradation by tissue enzymes. Their phlogogenic effects are apparently related to their ability to activate the alternate complement pathway (ACP). The in vitro activation of the ACP by lysozyme-treated cells and cell walls of group A, B, and D streptococci suggests that both rat and human lysozyme can modulate this activity, i.e., increasing it, decreasing it, or doing both in that order. The effects of the lysozymes also correlated with the degree to which they can unmask the aminosugar-reducing groups detectable in a given amount of cell wall, which suggests that partial depolymerization of the cell wall is critical for ACP activation. The effects of mutanolysin and C phage lysin on ACP activation were found to be correlated with their action on streptococcal cell walls. Neuraminidase had relatively little effect on ACP activation by most streptococcal strains tested. We conclude that the participation of tissue enzymes, including but not necessarily limited to lysozyme, is an important determinant for the clinical arthritis induced by group A, B, or D streptococci. Experimental arthritis induced in rats with whole (or disrupted) streptococci may depend both on the capacities of the cell walls to activate the ACP and on the capacities of the host tissue enzymes to modulate this activation. Great severity and long durations of the disease were determined by the capacity of the enzymes to degrade cell wall antigens to a degree sufficient to ensure efficient activation of the ACP without completely degrading the material so that it no longer activates complement. In this model, the limited resistance of group B peptidoglycan to lysozyme was a critical pathogenic factor.

Animals↗
PubMed1987

Susceptibility of pathogenic and nonpathogenic Naegleria spp. to complement-mediated lysis.

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with [3H]uridine and visual observation with a compound microscope were used as indices of lysis. Highly pathogenic mouse-passaged N. fowleri was less susceptible to the lytic effects of NHS and NGPS than the weakly pathogenic, axenically grown N. fowleri or N. australiensis and the nonpathogenic amoebae N. gruberi and N. lovaniensis. However, both pathogenic and nonpathogenic Naegleria spp. depleted complement as assessed by total hemolytic activity. Treatment of serum with EDTA, heat (56 degrees C, 30 min), cobra venom factor, or antibody to C3 or C9 complement components decreased the amoebicidal activity of NHS. The presence of specific agglutinating antibody to N. fowleri enhanced the amoebicidal activity of NGPS for N. fowleri.

Amebiasis↗
PubMed1987

Activation and binding of C3 by Candida albicans.

Interaction with components of the complement system is an important aspect of the pathogenesis of infection by Candida albicans. The key role of C3 as an opsonic ligand and as an element in amplification of complement activation led us to examine several factors that influence the activation and binding of C3 cleavage fragments to the yeast. Activation and binding of C3 were determined by use of normal human serum containing 125I-labeled C3. Incubation of yeast-phase cells in 20% serum led to deposition of 2.5 X 10(5) to 3.0 X 10(5) molecules of C3 per yeast cell. Binding of C3 was absent in serum that was heat inactivated for 30 min at 37 degrees C or in serum that was chelated with EDTA. Chelation of serum with EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] reduced binding of C3 fragments by 31%. These results suggest that the alternative complement pathway is the primary mechanism for activation and binding of C3 fragments to C. albicans. Bound C3 could be partially removed (50%) by treatment with 1.0 M hydroxylamine. In contrast, 1.0 M hydroxylamine removed 98% of the C3 fragments bound to encapsulated Cryptococcus neoformans. These results suggest that ester bonds are the primary mechanism for binding C3 to C. neoformans, whereas binding of C3 to C. albicans involves both ester and amide bonds. Monoclonal antibodies specific for C3c and an iC3b neoantigen were used to identify the fragment of C3 that was bound to C. albicans. The results showed that the primary fragment bound to the yeast was C3b. An examination of the kinetics of activation and binding of C3 fragments showed that activation and binding were very rapid. Near-maximal binding occurred after a 2.5- to 5-min incubation period. In contrast, activation and binding of C3 fragments to C. neoformans proceeded at a slower rate, with maximal binding requiring 10 to 20 min. These results indicate that activation and binding of C3 fragments by the yeasts C. albicans and C. neoformans differ in several important characteristics.

Antibodies, Monoclonal↗
PubMed1992

Interactions between conidia of Aspergillus fumigatus and human complement component C3.

Activation and deposition of C3 on Aspergillus fumigatus conidia have been previously demonstrated. This study investigated in further detail the interactions between complement component C3 and the conidia of A. fumigatus. Immunoblotting and 125I-C3 binding studies showed that C3 deposition was rapid (less than 15 min) and parallel to the formation of iC3b. Immunoblotting experiments identified a 54- to 58-kDa conidial protein which binds human complement component C3 and/or a C3 fragment(s). 125I labeling of the outer layer of the conidia demonstrated that this protein doublet was present on the surface of the spore. The further degradation of C3 to low-molecular-mass fragments (40, 37, and 30 kDa), in the absence of plasma, by intact living conidia and a preparation of the outer conidial wall layer indicated the ability of fungal components to cleave C3. These data suggest that interactions between conidia and C3 are not limited only to deposition via activation of the alternative complement pathway; they also include degradation of bound C3.

Animals↗
PubMed1993

Complement resistance of pathogenic Entamoeba histolytica mediated by trypsin-sensitive surface component(s).

Pathogenic forms of the protozoan parasite Entamoeba histolytica were reported previously to resist the cytolytic effect of the alternative complement pathway (AP) only temporarily during exposure to complement. In contrast, nonpathogenic forms of E. histolytica had been found to show AP resistance as a stable property. We studied the mechanisms of AP resistance of the two forms. Upon exposure to AP activity, resistant pathogenic or nonpathogenic forms bound significantly less C3 products than complement-sensitive pathogenic amebae, indicating that the two resistant forms both inhibited AP amplification. Various enzymatic treatments and inhibition of membrane mobility by cytochalasin B and glutaraldehyde fixation showed that the mechanisms of AP inhibition differed between pathogenic and nonpathogenic forms; in contrast to nonpathogenic forms, pathogenic amebae required intact membrane mobility and a trypsin-sensitive surface component(s) to inhibit AP activation.

Animals↗
PubMed1993

TnphoA-mediated disruption of K54 capsular polysaccharide genes in Escherichia coli confers serum sensitivity.

To assess whether non-K1, group 2 capsular serotypes are important in conferring serum resistance to extraintestinal isolates of Escherichia coli, a K54 blood isolate (CP9) was evaluated as a model pathogen. Transposon mutagenesis (TnphoA) was used to generate isogenic capsule-negative mutants. CP9 was resistant to the bactericidal effects of serum, growing in 80% serum. In contrast, all of the capsule-negative mutants had an increased sensitivity to 80% normal human serum, undergoing a 2- to 3-log kill over 3 h when starting inocula of 10(4) to 10(7) CFU/ml were used. The killing of the capsule-negative strains was mediated through the alternative complement pathway and not by lysozyme or beta-lysins. The protective effect of the K54 capsule against the bactericidal activity of serum was not through inhibition of the complement cascade, nor did it appear to be through a difference in the binding of C3.

Antimicrobial Cationic Peptides↗
PubMed2004

Biological activities of naturally occurring antibodies reactive with Candida albicans mannan.

Sera from normal adult humans may contain high levels of antibody reactive with Candida albicans mannan. This study examined selected biological activities of such antibodies, focusing on sera that were collected from 34 donors and analyzed individually. The results showed that antimannan titers were normally distributed. Reactivity as determined by enzyme-linked immunosorbent assay with serotype A mannan generally paralleled reactivity with serotype B. Analysis of the kinetics for activation of the complement system and deposition of complement component 3 (C3) onto serotype A and serotype B cells showed a decrease in the lag time that occurred before the onset of rapid accumulation of C3 that correlated with increasing antimannan titers. In contrast, there was a decrease in the overall rate of accumulation of C3 on serotype A cells that was strongly correlated with increasing antibody titers; serotype B cells showed no such decrease. An evaluation of the contribution of mannan antibody to opsonophagocytic killing showed that mannan antibody in individual sera and antimannan immunoglobulin G (IgG) affinity purified from human plasma contributed to killing by neutrophils in a dose-dependent fashion in the absence of a functional complement system. However, affinity-purified antibody in very high concentrations was inhibitory to both complement-dependent and complement-independent opsonophagocytosis, and this finding suggests a prozone-like effect. In contrast, if the complement system was functional, antimannan IgG was not needed for opsonophagocytic killing. These results suggest that naturally occurring mannan antibodies and the complement system are functionally redundant for opsonophagocytic killing by neutrophils.

Adult↗
PubMed2006

Herpes simplex virus type 1 and 2 glycoprotein C prevents complement-mediated neutralization induced by natural immunoglobulin M antibody.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.

Animals↗
PubMed1999

A novel assay for serum complement activity: C42 generation assay.

BACKGROUND: In most clinics, laboratory tests for serum complement are limited to immunochemical determinations of C3 and C4 and are occasionally extended to the hemolytic titration of total complement functional activity (CH50). However, these tests are often not sufficient for the analysis of low CH50 serum. METHODS: A novel assay for serum complement activity, the C42 generation assay, has been developed. The principle of this assay is based on the hemolysis of sensitized sheep erythrocytes (EA) by complement components in two sera: C42 (the classical pathway C3 convertase) is generated on EA by C1, C4 and C2 in the first serum, followed by a second reaction leading to hemolysis by C3-C9 supplied by the addition of the second serum in the presence of EDTA. RESULTS: This methodology permits the evaluation of two distinct serum complement activities of a test serum. The combined activity of C1, C4 and C2, as well as the combined activity of C3-C9, can be estimated from the observed degrees of hemolysis. Information obtained from this assay is helpful for the analysis of test serum determined to have decreased CH50. Several clinical cases are presented in which this assay was utilized. CONCLUSIONS: The C42 generation assay is another functional assay of serum complement which can provide information beyond that obtained from the typical serum CH50 assay. Since intermediate cells or isolated complement components are not necessary, this assay can be employed rapidly and economically in a clinical setting.

Animals↗
PubMed1988

Discrepancy between effects of in vivo and in vitro administration of gammaglobulin on phagocytic killing of Streptococcus pneumoniae in an antibody-deficient serum.

Phagocytic killing of Streptococcus pneumoniae serotypes 6A, 14, 18C, 19F and 23F was investigated in the dysgammaglobulinemic serum of a patient with recurrent pneumococcal infections. Previous studies with this serum had established combined IgG2, IgG4 and IgA deficiency, deficiency with regard to specific antipolysaccharide antibodies and essentially normal complement functions. Phagocytic killing of all serotypes was reduced in the patient's serum. Addition of immunoglobulin in vitro enhanced both classical and alternative complement pathway mediated opsonization. In constitution experiments neither purified Clq nor CRP influenced phagocytic killing. Surprisingly, intramuscular administration of a fairly small dose of gammaglobulin to the patient was associated with a rapid increase in the serum opsonic activity for serotype 23F. The increased opsonization occurred before specific anticapsular antibodies were detectable in serum. The findings suggest that the possible effects of gammaglobulin treatment may not exclusively be related to the acquisition of serotype-specific antibodies.

Complement Activating Enzymes↗